One-step visualization of natural cell activities in non-labeled living spheroids

3D cultured cell aggregates, including spheroids, reflect the gene expression patterns of living tissues/organs. Mass preparation of spheroids enables high-throughput drug screening (HTS). However, conventional optical imaging of spheroids makes it difficult to obtain sufficient resolution of individual living cells in the thick cellular stack. Rapid and accurate assessment of cellular responses in spheroids is required for effective drug screening. Here, we show that negative contrast imaging (NCI) of spheroids overcomes this issue. Hydrophilic fluorescent dye added into the culture medium rapidly diffused into the intercellular space of living spheroids within a few minutes. Confocal microscopy showed the NCI of individual cells as dark and detailed contours clearly separated with fluorescence signals in the intercellular space. NCI enables the visualization of the alteration of cell morphology after anti-tumor drug application to living spheroids and the measurement of the fluorescent dye diffusion rate without any complicated pretreatments. Using this system, we found that the antitumor drug doxorubicin reduced the intercellular space of spheroids consisting of the human hepatocyte carcinoma cell line HepG2, through the activation of TGF-β signaling and upregulation of ECM protein expression, implicating a drug resistance mechanism. Collectively, the combination of NCI of spheroids and HTS may enhance the efficiency of drug discovery.


Result
Evaluation of NCI for instant observation. NCI observes a negative contrast image of a target of interest. The target structure in fluorescent dye-filled space excludes the surrounding fluorescent dye by its volume; thus, confocal imaging visualizes the weak (or no) fluorescence area, such as the target's shadow (Fig. 1a). NCI has been utilized for organelle visualization 9 and the brain 10 without any specific labeling of the target. We hypothesized that the addition of a hydrophilic fluorescent dye into the medium could aid the visualization of the living cells in the spheroid by NCI. We added a commercially available hydrophilic fluorescent dye, Alexa Fluor (AF) 488-NHS (MW, 643.4 Da), to the medium in which HepG2 spheroids were cultured. The dye immediately diffused into the intercellular space of the living spheroid cells and became visible within two minutes, but not in the cytoplasm of cells. The individual living cell bodies with weak fluorescent signal in the intercellular space were outlined in detail by confocal microscopy (Fig. 1b left). While other hydrophilic fluorescent dyes including AF647 (MW, 1025.2 Da) also gave similar confocal images (Fig. 1c,d), hydrophobic dyes showed predominant accumulation in the peripheral cells of the spheroids (Extended Data Fig. 1, Rhodamine 6G (Rh6G) and 11 ). We then examined the NCI of spheroids with HEK293T cells expressing membrane-anchored AcGFP. The AcGFP signal and the external AF647 signal were well aligned (Extended Data Fig. 2). This indicated that the diffused AF647 signal clearly marked the extracellular space of the spheroid. Furthermore, AF488conjugated albumin (MW, 66.5 kDa) took a longer time to diffuse into the intercellular space (Extended Data Fig. 3). We concluded that small (~ 1000 Da of MW) hydrophilic dyes are required for NCI of spheroids. Under these conditions, we could identify the individual spheroid cells located at a depth of 100 μm from the adhesion plane (Extended Data Fig. 4).
Next, we compared the performance of NCI with that of conventional imaging methods for living cells. One membrane-permeable fluorescent dye, SiR-act, can stain the cytoskeleton of living monolayered cells 12 . We used multicolor confocal microscopy of SiR-act as far-red fluorescence and AF488 for NCI as green fluorescence on a single HepG2 spheroid. AF488 signals diffused into the spheroid core within 5 min with clear visibility of individual cellular structure (Fig. 1b, upper left). In contrast, SiR-act required several hours to show clear fluorescent signals, with little fluorescence at the core region (Fig. 1b, lower right). These data showed that NCI significantly improved the performance of imaging, offering a rapid and deep field of view for individual spheroid cells, compared to conventional live cell imaging methods.
Evaluation of NCI for long-term observation. NCI can also enable the long-term observation of living spheroids. Time course observation of single spheroids at 30 min intervals with a single AF647 addition at the starting point showed cytokinesis of the cells in the spheroid core. The spherical parent cell (Fig. 1c, left, dashed rectangle, and inset) was divided into two daughter cells (Fig. 1c, right, dashed rectangle, and inset). Sequential confocal tomograms gave the 3D structure and cell volume, and the daughter cells (Fig. 1c right, lower inset, 1066 and 1165 μm 3 ) were accurately measured to be one-half the volume of their parent cell (Fig. 1c left, lower inset, 2211 μm 3 ). An hour after dye addition, we also observed punctate small fluorescent spots within the spheroid core cells, indicating endocytosis ( Fig. 1d right, arrowhead). Spheroids cultured for long periods expand in size, and the core cells begin necrosing due to poor nutrition and oxygen 5 . The NCI of day 16 HepG2 spheroids showed fluorescence accumulation in the core region that partially merged with signal from the cellular necrosis indicator propidium iodide (PI) (Fig. 1e, asterisk), and clear separation of individual cells in the peripheral region, stained with the living cell indicator calcein, was evident (Fig. 1e, sharp). This indicates that hydrophilic dye accumulation in spheroids can also be used as a cell death indicator in NCI.
Interestingly, NCI also visualized contaminated noncellular components that can clearly be distinguished from spheroid cells. Such components often existed in the medium, and likely come from the serum and/or culture device; sometimes the cells assemble on the debris (Extended Data Fig. 5, arrowhead). This suggested Figure 1. NCI visualizes the structure and activity of individual living cells in the spheroid core. (a) Schematic imaging principle of spheroid NCI. Diffusion of the AlexaFluor (AF) fluorescent dye into the spheroid is observed by confocal microscopy. Individual living cells in the spheroid are visualized as a dark shadow of the cell contour. (b) Comparison of the two confocal images of the same HepG2 spheroid visualized by NCI (AF488) and by the representative conventional living cell staining method, SiR-actin (SiR), at 5 min and 3 h after dye addition into the medium. Bar: 100 µm. Arrowheads: bile canaliculus-like structures. Inset: Bright field. Bar: 100 µm. (c) Visualization of cytokinesis of a single representative HepG2 spheroid cell unfolding over time. The spheroid incubated at 37 °C and 5% CO 2 in a humidified incubator on the stage of the microscope was scanned at 0.5-h intervals. The AF647 was added once at the first NCI acquisition. Bar: 100 µm. Upper inset: enlarged image of the dashed rectangle. Arrowhead: the parent-and daughter cell(s). Lower inset: reconstructed 3D structure of the target cell(s). Bar: 20 µm.  www.nature.com/scientificreports/ width of the intercellular space. Cell aggregates are regarded as a potential model to measure the drug diffusion rate in tissue. We measured the diffusion rate of AF647 using NCI. Before the addition of AF647 into the spheroid medium, we undertook NCI, using AF488, and manipulated the scanning area and the distance from the adhesion plane (0, 25, 50, and 75 μm) (Fig. 2a, left column). To observe saturation of dye diffusion, 30 s after addition of AF647 into the medium, confocal images were acquired at 30 s intervals for 10 min. The intensity gain of AF647 fluorescence at the spheroid core was saturated within 4 min (Fig. 2b). To calculate the diffusion rate, time course image acquisition in short periodic intervals was performed ( Fig. 2a right, c). The diffusion rate from the linear approximation curve created within 6 to 26 s was 293.98 s −1 . These data demonstrated the ability of NCI to measure the diffusion rate of various compounds into the spheroid, although even if we have to do fluorescent labeling. Combined with the endocytosis observations ( Fig. 1d), this method can measure the diffusion rate of drug transport from the outside into the cytoplasm of the spheroid cells.
Distinct NCI patterns for spheroids of different cell lines. Spheroids are now regarded as a standard model for HTS. We evaluated whether the NCI of spheroids was suitable for HTS imaging. We first examined whether NCI could reveal distinct characteristics of different cell lines, including HEK293T, HepG2, and the human colon cancer-derived cell line HCT116. We found clear differences in the cellular filling patterns of these cell lines. The filling pattern of HepG2 spheroids showed a compacted appearance resembling paving stones together with bile canaliculus-like structures (Fig. 1b, arrowhead 13,14 ), compared with the relatively sparse cellular filling of the HEK293T spheroid ( Fig. 3a left). The HEK293T spheroids showed Rosetta structures within the core (Fig. 3a, arrowhead), which likely represent vigorous cell proliferation. Interestingly, the HCT116 spheroids showed a biphasic structure with a compressed core and sparse periphery (Extended Data  www.nature.com/scientificreports/ We next examined whether NCI can identify the effects of culture conditions and culture times on spheroid structure and viability. We compared two culture conditions, the low-adhesion plate used in the present study, and the hanging drop method. After 5 days of incubation, the spheroids under these two conditions showed little difference in appearance, viability, and individual cellular volume (Extended Data Fig. 7a). In contrast, after 12 days of incubation, the spheroids from the low-adhesion plate showed marked cell death compared with the hanging-drop method (Extended Data Fig. 7b-d). Additionally, the histogram of the cellular volume was clearly less in the low-adhesion plate spheroids, than in the hanging drops (Extended Data Fig. 7e). Enlargement of spheroids may cause insufficient medium diffusion, hypoxia, and low nutrition in the core region, causing both cell differentiation and cell death. Thus, NCI can show various characteristics of spheroid cells, including cell death and differentiation, supporting its use for HTS.
Alteration of spheroidal structure with anti-tumor drugs. Next, we attempted to observe the drug response of spheroids by NCI. HEK293T spheroids were treated overnight (18 h) with the two antitumor drugs, the multifunctional antitumor drug doxorubicin (DX) and microtubule stabilizer paclitaxel (PX). Both of the drugs conferred only slight changes in the whole spheroid size observed by conventional optical imaging ( Fig. 3a-c, insert), but there was a clear difference in the cellular filling structure by NCI. DX treatment eliminated the Rosetta structure observed in non-treated spheroids (Fig. 3a, arrowhead), and induced tight cellular www.nature.com/scientificreports/ fillings (Fig. 3b). This may be due to the volume increase of the cells from DX, which arrests cells in G2 phase. In contrast, PX treatment induced expansion of the intercellular space together with a morphological change of the cells to a spherical shape (Fig. 3c), probably due to the inhibition of microtubule turnover. These changes were also apparent in a quantitative fashion in terms of a change in the intercellular space (Fig. 3d), the individual cell contour ( Fig. 3e-g) and the 3D structure (Fig. 3h). To our knowledge, this is the first report of such druginduced structural changes in spheroid cells.

Doxorubicin-induced ECM expression in spheroids of HepG2 cells.
The DX-treated HepG2 spheroids showed a more intriguing phenomenon. Non-treated HepG2 spheroids showed clear and compacted intercellular spaces in the core region (Figs. 1b and 4a), indicating smooth diffusion of the dye into the space. However, DX treatment reduced the fluorescence intensity in the core region, and produced a less clear intercellular space (Fig. 4b).
To uncover the concerning genes, we undertook RNAseq analyzing against the 4 experimental group designated by culture style (monolayer culture or spheroid) and by with/without DX treatment. The transcriptomic analysis of the DX-treated spheroids showed clear difference of gene expression pattern among the experiment group (Extended Data Fig. 8c, S9, S10) and the activity of several gene function pathways including the TGF-β signal cascade (Extended Data Fig. 8d, asterisk), as well as the upregulation of some ECM components, including collagens and fibronectin (Table 1 and Extended Data Fig. 11). Although such the upregulation of the genes were not specific on spheroid, we simultaneously applied DX and the TGF receptor specific inhibitor LY36497 to determine whether the TGF-β signaling cascade is associated with the decrease in fluorescence intensity. As expected, co-administration of LY36497 reduced the effect of DX (Fig. 4d,f), and also suppressed DX-induced fibronectin expression ( Fig. 4g and Extended Data Figs. S12, S13). These results suggest that DX may promote the expression of ECM proteins through the TGF-β signaling cascade to reduce the AF diffusion efficacy by upregulating ECM density.

Discussion
In the present study, we demonstrated the utility of NCI in living spheroid cell imaging. Simple addition of the hydrophilic fluorescent dye into the medium provided quite dramatic results, within a few minutes, for the confocal image, with well-separated individual cell bodies in the core region of the spheroids. Image analysis provided quantitative results, such as individual cell volume. This minimally invasive imaging method revealed antitumor drug-induced changes in cell structure and the molecular diffusion of drug into the intercellular space, which can be used to immediately evaluate drug effects.  We demonstrated that NCI can detect detailed shapes of individual cells of the spheroid core without invasive manipulation, which represents a breakthrough overcoming technical limitations of more conventional methods. By observing spheroids with NCI, we were able to visualize drug responses of the spheroids in terms of structural (Fig. 3) and dye-diffusion characteristics (Fig. 4), differences in cell lines (Fig. 1, 3, and Extended Fig. 6), cellular viability ( Fig. 1 e), cellular functions such as cytokinesis (Fig. 1c) and endocytosis (Fig. 1d) and non-cellular components (Extended Fig. 5). This was all possible with the simple addition of hydrophilic fluorescent dye to the medium for just a few minutes. These advantages were mainly due to the rapid inflow diffusions of the dyes we used (Fig. 2), compared with dyes used in previous studies [15][16][17] . The significant expansion in parameters that one can visualize, as well as the shortening of the observation time and the simplification of the process, should facilitate studies of drugs of interest using this model. At the same time, single cell-level observation can minimize the impact of spheroid variation, and thereby facilitate quality control. Of note, these other methods can only utilize single wavelengths, whereas in the present study, we utilized a total of three wavelengths for fluorescent imaging (Fig. 1e) as well as a bright field image. This suggests that NCI can improve confocal microscopy, and simultaneously facilitate both HTS and basic research. In combination with the molecular imaging of specific genes, NCI can provide structural data at the single-cell level, visualize the response of individual cells to drugs of interest, and enhance the progress of drug discovery. Further, non-cellular components can be observed by NCI (Extended Fig. 5), but not by conventional living cell imaging methods. The ability to identify non-cellular components for checking and controlling the quality of spheroids should also be helpful for drug discovery. NCI may also be suitable for the study of cellular/noncellular components, such as bone, cartilage, and artificial materials, and may be important for the field of regenerative medicine in the future.
We also demonstrated that NCI can be useful for the evaluation of the response of the spheroid to certain drugs, with a readout of an alteration of dye diffusion into the spheroid core. We have highlighted the effect of DX on the hepatoma cell line spheroids (Fig. 4). DX has multiple clinical effects such as apoptosis induction by accumulation in mitochondria, ROS generation and inhibition of transcription 18 , and we surely detected upregulation of ribosomal proteins (Extended Data Figs. 9, 10, asterisk). That is therefore regarded as a first choice for treatment of some tumors 19 . However, longitudinal administration of DX often causes fibrosis in healthy tissues, including the liver 20 and cardiovascular endothelium 21 . Furthermore, tumor fibrosis caused by antitumor drugs, including DX, is now a serious concern because it reduces the transport efficiency of the drug into the affected area 22 , thus demanding the simultaneous application of anti-fibrosis drugs 23 . Using NCI, we demonstrated a correlation between DX stress on spheroid cells and the reduction of the dye diffusion rate into the spheroid core due to increased expression of ECM proteins. It has also been suggested that ECM upregulation is triggered by the TGF-β signaling cascade 24 . We confirmed this by using a specific inhibitor of the TGF-β receptor and observing recovery of the dye diffusion rate into the spheroid core (Fig. 4). Of note, the result indicated that the NCI-based spheroid assay could be utilized both for evaluation of drug-induced ECM augmentation causing tissue fibrosis, and as a screening assay for drug candidates that suppress fibrosis. Overall, our results demonstrated the utility of NCI for HTS drug screening.
There remain two tasks for the actual application of NCI to HTS. The first is the difference in the degree of cell packaging among the cell types that affects dye diffusion into the spheroid core. As shown by the HCT116 spheroid in Extended Data Fig. 6, tightly packaged spheroids had lower dye diffusion rates and less intense fluorescence signals in the intercellular space. The affinity of cell-cell adhesions, such as in tight junction formation, may affect the dye diffusion rate. Together with the ability to observe deep inside the spheroid, as shown in Extended Data Fig. 4c, the image sharpening and ability to detect weak fluorescent signals may overcome this issue. The other task is to automate image processing and analysis. The NCI of spheroids provides a significant amount of information in a single spheroid study. To ensure reproducibility and shorten analytical time, automatic analysis should be performed, especially to translate drug-induced structural changes to the study of drug mechanisms.
In conclusion, we have developed a simple and effective live imaging method, NCI, which can measure many parameters of individual cells in the spheroid core region. Simple and rapid visualization with low invasiveness should be suitable for studies utilizing primary cultured cells and stem cells that are difficult to manipulate genetically. NCI also uncovered the cell line-dependent cell packing pattern and unique structural changes in response to anti-tumor drugs. Transcriptomic analysis of conditioned spheroids provided gene candidates corresponding
Cell culture. The HepG2 human hepatoma cell line (JCRB1054) and the HCT-116 human colon cancer cell line (JCRB1408, stably expressing luciferase) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) and tested for mycoplasma contamination. The HEK293T human embryonic kidney cell line was described previously 25 . Cells were cultured at 37 °C and 5% CO 2 in a humidified incubator in RPMI-1640 supplemented with l-glutamine and phenol red supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). HEK293T cells stably expressing plasma membrane-anchoring AcGFP were prepared with pAcGFP1-Mem transfection and selection using G418 resistance. Western blotting. Unless otherwise stated, the following process was performed at room temperature (25-30 °C). A total of 40 targeted spheroids were collected into one 1.5 mL low-adhesion microtubes using a pipette. The medium was carefully removed, and spheroids were washed twice with Tyrode's buffer containing phenylmethylsulfonyl fluoride (Fujifilm) and protease inhibitor cocktail (Sigma). The pellet was suspended in 40 µL of 1 × sample buffer, sonicated for 15 min at 4 °C, and incubated at 100 °C for 5 min. A 15 µL sample of the supernatant was subjected to SDS polyacrylamide gel electrophoresis (PAGE, 8% acrylamide gel, 75 min at 30 mA constant current), and the gel was electroblotted onto a Immobilon FL membrane (Millipore) for 90 min at 13 V. The membrane was then blocked in Tris-buffered saline (TBS) buffer with 1% (w/v) skim milk at 4 °C overnight with gentle shaking. The membrane was then incubated in 1% (w/v) skim milk-0.1% Tween20containing TBS buffer with anti-FN1 antibody (final concentration: 2 µg/mL) and incubated for 1 h. The membrane was washed and suspended in 1% (w/v) skim milk-0.1% Tween20-0.1% SDS-containing TBS buffer with IRDye800-labeled anti-rat Ig antibody solution (final concentration: 0.1%) for 45 min. After three washes with 0.1% Tween20-containing TBS buffer and once with TBS buffer, the membrane was dried, and the fluorescence signals were scanned using an Odyssey system (LI-COR, Lincoln, NB, USA).
Immunofluorescence. Five targeted spheroids were pipetted into one 1.5 mL low-adhesion microtube. www.nature.com/scientificreports/ 30 µL sample of the supernatant was added to 300 µL of spheroid-containing medium by gently pipetting several times. The dish was then incubated in a humidified incubator for the required time at 37 °C and 5% CO 2 until microscopic observation. To compare the living cell imaging ability of NCI and SiR-actin staining, SiR-actin (final concentration: 3.3 µM), verapamil (10 µM), and 30 µL of AF488 were simultaneously added to 300 µL of the medium. The prepared spheroids in the glass-bottomed dish were imaged on a Zeiss LSM510 or LSM710 confocal microscope with a C-Apochromat 40 × /1.2 W water immersion lens and ZEN software (Zeiss, Jena, Germany). The cells were visualized without changing the medium to avoid stress or morphological changes in the cells. Confocal images were acquired at a distance of 50 µm from the adhesion plane of the spheroid. Two images were acquired for averaging at a pixel size of 512 × 512 (scaling: 0.415 × 0.415 µm) with 1.0 µm pinhole diameter as the software-recommended scan speed. AF488, calcein, and AcGFP were excited at 488 nm using an argon laser at a power of 2.0%. Red fluorescent dyes, PI and Rh6G, were excited at 543 nm using a helium/neon laser at 2.0% power. AF647 and SiR-actin were excited at 633 nm using a helium/neon laser at 2.0% power. Serial confocal images for z stacking were acquired at 1.0 µm intervals starting at the adhesion plane of 0 µm.
For cell viability imaging shown in Fig. 1e, calcein (final concentration: 3.3 µM) and PI (1.6 µg/mL) were added to the medium and incubated 18 h prior to imaging, after which 30 µL of AF488 was added before imaging. The prepared spheroids in the glass-bottomed dish were imaged on a Zeiss LSM710 confocal microscope with EC Plan-Neofluar 20 × /0.50 M27 correction and ZEN software (Zeiss). Confocal images were acquired at a distance of 75 µm from the adhesion plane of the spheroid. Sixteen images were acquired for averaging at a pixel size of 1024 × 1024 (scaling: 0.415 × 0.415 µm).

Measurement of AF diffusion rate.
Before measurement, the prepared spheroids were transferred into 8 well LabTek chambered cover glasses with 300 µL of the medium and incubated for 3 h at 37 °C and 5% CO 2 for adhesion of the spheroid on the chamber. The chamber was then set on the microscope stage on an LSM710 confocal microscope with a C-Apochromat 40 × /1.2 W correction and ZEN software (Zeiss). First, for the z position setting, 20 µL of AF488 stock solution was mixed into the chamber. After 5 min, the z-position of the spheroid was adjusted by NCI excitation at 488 nm. Then, 100 µL of the medium was removed from the chamber and 30 µL of AF647 stock solution was mixed and returned to the chamber at the start of the measurement. For the long periodic observation shown in Fig. 2b, the first confocal image set was acquired after 10 s of dye addition, and then acquired sequentially at 30 s intervals over 10 min. The confocal images were acquired at a position below the adhesion plane of 0-100 µm with a 10 µm interval, a size of 512 × 512 nm 2 , and a gain of 690 with no averaging.
For the short periodic observation shown in Fig. 2c, the first confocal images were acquired after 6 s of dye addition, and then acquired sequentially at 4 s interval over 50 s. Confocal images were acquired at 0, 25, 50, and 75 µm from the adhesion plane. The images were sized at 256 × 256 nm 2 , gain of 690, and pinhole of 1.0 AU (90 µm diameter for AF647) with no averaging.
Image processing and analysis. All images were prepared using Zen software (Zeiss) by adjusting the gain and black/white balance. Structural analysis of the individual cells was performed using Neurolucida (MBF Bioscience, Williston, VT, US). Individual cell contours were picked semi-automatically from the single NCI image by Neurolucida, and the structural parameters (area, perimeter, and roundness) were calculated (Neurolucida, Figs. 1f-h, 3e-g, and Extended data Fig. 6b-d). The cell number are around 150 contour of cells per single confocal image. The histogram of the structural parameters was prepared using Excel software. The reconstructed 3D cell structures were prepared using Neurolucida from the sequential confocal tomogram from 0 to 100 µm with 2 µm interval acquisition (Fig. 1c,i insert, Fig. 3a-c insert, and Extended Data Fig. 6e). The histogram of cell volume in Figs. 1i, 3h and Extended Data Fig. 6f were prepared with the reconstructed 3D cells located in the 50 × 50 × 100 µm virtual area at the center of the scanned spheroid. The cell number were around 50 per analyzed area in single spheroid. Fluorescence intensities were analyzed using Fiji software (https:// imagej. net/ Fiji). The dye diffusion rate graph was prepared from the fluorescent intensity of the 50 × 50 µm virtual area at the center of the NCI image scanned at 70 µm (Fig. 2b) or 75 µm (Fig. 2c) above the adhesion plane. The drug-response change of the intercellular space shown in Fig. 3d was calculated by Fiji by subtracting the whole spheroid area of a single NCI image from the intercellular space area, and both were defined by automatic threshold setting of Fiji. The DX response fluorescence change in Fig. 4f was calculated by comparing the total fluorescence of the spheroid area in the target NCI image to the non-treated sample, set at 100%. The histogram indicating fluorescence change was obtained across the intercellular space of the spheroid and visualized with AF and the plasma membrane with AcGFP, which was temporally expressed in the cytoplasm conjugated with plasma membrane transporting signal peptide (Extended data Fig. 2); it was plotted from a single LSM image of the HEK293T spheroid and quantified using Zen software (Zeiss).

RNA preparation.
A total of 180 spheroids with or without DX treatment were collected and washed twice with PBS. The spheroids were transferred to low-adhesion microtubes and homogenized using a QIAshredder (QIAGEN, Hilden, Germany). Total RNA was harvested using an RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's protocol. The total RNA bound to the spin column was treated with recombinant DNase I (30 units, TOYOBO, Osaka, Japan) for on-column digestion for 15 min at room temperature and washed again with the accessory solution. Total RNA was eluted with 50 µL of MilliQ water, and its RNA concentration and quality were assessed by reading the absorbance at 230, 260, and 280 nm. Approximately 5 µg of total RNA was collected using this protocol.
In brief, mRNA was enriched from the total RNA sample, fragmented, double-stranded cDNA synthesized, which was ligated to adaptor sets, enriched by polymerase chain reaction, and sequenced on an Illumina HiSeq (San Diego, CA, USA). Each dataset was subjected to filtering, human genome mapping, quality check, differential gene expression analysis, and gene ontology enrichment analysis (KEGG).
Statistical analysis. The number of replicates is shown in the figure legends. Statistical significance was determined by one-way ANOVA followed by Dunnett's test or the Tukey-Kramer test, and the P-values are shown as a respective graph. PRISM version 9.2.0 (GraphPad Software) was used for the analysis. Due to the small sample size of each experimental condition, we could not perform normality test. One-way ANOVA was performed assuming a normal distribution.

Data availability
All data obtained during study are available from the corresponding author upon request.